Protein synthesis is required for optimal induction of 25-hydroxyvitamin D3-24-hydroxylase, osteocalcin, and osteopontin mRNA by 1,25-dihydroxyvitamin D3

The regulation of the 25-hydroxyvitamin D3-24-hydroxylase gene by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been extensively studied. It is well established that two vitamin D response elements in the promoter are responsible for the 1,25(OH)2D3 induction of transcription. Surprisingly, this induction is blocked by the protein synthesis inhibitor, cycloheximide (CHX). In AOK-B50 cells, 1,25(OH)2D3 caused a large induction of 24-hydroxylase mRNA by 7 h; however, the addition of CHX simultaneously or 2 h after 1,25(OH)2D3 addition caused 76.4±13.0 and 37.1±18.8% reductions in the mRNA, respectively. Addition of CHX 4 h after 1,25(OH)2D3 had the opposite effect, and 21.7±17.2% more mRNA was observed after 7 h. Similar patterns of mRNA expression were observed in other cell lines. CHX also decreased the induction by 1,25(OH)2D3 of osteocalcin and osteopontin mRNA in ROS17/2.8 cells when added together with 1,25(OH)2D3. The effect of CHX on the expression of a stably transfected luciferase construct under the control of 1400 bp of 24-hydroxylase promoter indicates that a 1,25(OH)2D3-inducible transcription factor(s) that acts in the promoter region may at least in part be responsible for the effect of CHX on mRNA production of target genes.